Cervicovaginal specimen biomarkers for early detection of ovarian and endometrial cancer: A review

Abstract Background In the last decade, technical innovations have resulted in the development of several minimally invasive diagnostic cancer tools. Within women at high risk of developing ovarian or endometrial cancer (EC) due to hereditary cancer syndrome, there is an urgent need for minimally invasive and patient‐friendly methods to detect ovarian cancer and EC at an early stage. Materials and Methods We performed a systematic search of studies using DNA methylation or mutation analysis, microbiome, or proteomics performed on cervicovaginal specimens (smear, swab, or tampon) intended to detect ovarian and EC published until January 2024. Results Included studies (n = 36) showed high heterogeneity in terms of biomarkers used and outcomes, and only a few studies reported on the detection of biomarkers in high‐risk subgroups. Conclusion Based on the findings in this review, DNA methylation techniques seem to be the most promising for detecting ovarian and EC at early stages in the general population. Future validation of cervicovaginal DNA methylation techniques is needed to determine whether this technique might be beneficial in hereditary high‐risk subgroups.

been proven to reduce cancer mortality and are therefore not recommended. 6,7omen who are at high risk of developing OC or EC, for example due to a hereditary cancer syndrome, may benefit from specific surveillance or risk-reducing strategies.Women with a BRCA1 or BRCA2 pathogenic variant have a cumulative risk of developing OC at the age of 80 years of respectively 44% and 17%. 8Surveillance with transvaginal ultrasound and CA-125 antigen detection in blood samples to detect OC at an early stage in this group has proven to be ineffective. 6,9,10Therefore, the only strategy to reduce OC-related mortality in BRCA pathogenic variant carriers is a preventive risk-reducing bilateral salpingo-oophorectomy (RRSO) around the age of 40, which can lead to short-and long-term morbidity caused by early surgical menopause. 11,12ynch syndrome is an autosomal dominant hereditary cancer syndrome caused by pathogenic variants in multiple DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2) or by a deletion in the EPCAM gene resulting in a loss of expression of MSH2. 13Women with Lynch syndrome are at an increased risk of developing both OC and EC.5][16][17][18][19][20] Additionally, MLH1, MSH2, and MSH6 mutations are associated with an increased lifetime risk of OC depending on the specific mutation between 1% and 38%, compared to approximately 1.5% in the general population. 14,17,21,22Women with Lynch syndrome can opt for annual surveillance, including transvaginal ultrasound with subsequent endometrial biopsy, or consider preventive hysterectomy, including RRSO. 23owever, evidence of the effectiveness of annual endometrial surveillance is contradictory, and the procedure is often painful for these women. 24omen with PTEN Hamartoma Tumor Syndrome (PHTS), which is caused by mutations in the PTEN tumor suppressor gene, have a lifetime risk of developing EC between 19% and 28%. 25 Surveillance strategies are expert-opinion based, as no evidence-based surveillance guidelines are available. 26here is a clinical need for simple and patient-friendly methods to detect OC and EC at early stages.In 2013, Kinde  et al. were the first to show that mutations in OC and EC can be detected in DNA from cervical smears, suggesting that cervical smears could be used for OC and EC detection. 27Other techniques, including analyses of the microbiome, epigenome, and proteome, followed.In this review, we provide an overview of studies on cervicovaginal specimen biomarkers for the early detection of OC and EC.We aim to identify the tests with the most clinical potential that might have a future role in OC and EC detection in women with a hereditary risk of developing OC and EC.

| Search strategy
A systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines was performed.This review was registered in the International Prospective Register of Systematic Reviews (PROSPERO, number 377155).An electronic literature search for eligible studies was performed in PubMed, Medline and the Cochrane Library.Articles published between January 2010 and January 2024 were included.The following search string was used on the 8th of January 2024: ((Endometrial Neoplasms

| Study selection
Two independently working authors (KK, VL) screened the articles for relevant titles and abstracts and subsequently full texts using the web application Covidence.When no consensus was reached between the two authors, discrepancies were resolved by consulting a third author (JP).
The included articles needed to be available in full text and written in English and had to report on the detection of OC and/or EC using DNA/RNA methylation or mutation, proteomics, or microbiome.The analyzed material needed to originate from cervicovaginal specimens obtained with a smear, swab, or tampon.Only papers with a minimum of 10 patients were included.Articles matching any of the following criteria were excluded from this review: other screening methods, specimens obtained from nonviable organs, case reports, (systematic) reviews, and studies not specifying the number of participants or methods used (defined as unspecified study).

| RESULTS
The search resulted in 448 records, which were all screened on title and abstract.A total of 82 articles were assessed as possibly relevant and were selected for full-text review.After full-text review, 46 articles were excluded for the following reasons: other study population or techniques (n = 28), unspecified study (n = 11), no full-text (in English) available (n = 6) and not enough included patients (n = 1).Hence, a total of 36 articles were included in the final review.Twenty-three studies reported on DNA methylation and/or mutation, seven on the microbiome, and six on proteomics.No studies have reported on RNA methylation.A flowchart of the literature search is shown in Figure 1.

| DNA methylation and mutation analysis
A total of 23 studies performed methylation or mutation analysis of DNA isolated from cervicovaginal smears, swabs, or tampons.Twelve studies reported on DNA methylation analysis, nine on DNA mutation analysis, and two on both techniques.An overview of all studies, including the specifications of the techniques and study population, is given in Table 1.The largest study cohorts are briefly summarized below.
Bakkum-Gamez et al. used a panel of 28 tampon-based methylated DNA markers to detect EC with promising results in 192 women, including 100 patients with EC.The panel of 28 methylated genes resulted in a 96% specificity and 82% sensitivity (AUC 0.91).A post-hoc analysis of a panel of three genes yielded AUCs comparable to those of the 28-panel. 29arrett et al. analyzed CpG island methylation profiles in cervical smears from a large cohort of women

T A B L E 1 (Continued)
(n = 786) to detect OC and EC, 33 resulting in AUCs of 0.76 and 0.81 for respectively.To assess whether this test is also informative in healthy women at high risk of developing OC, the association between methylation and the risk of developing OC in a subgroup of healthy BRCA1 pathogenic variant carriers was evaluated, resulting in an AUC of 0.62.In a follow-up validation study, a test using cervical smears for early detection of EC based on DNA methylation at 500 CpG sites was evaluated. 30After validating this test in a set of 64 EC patients and 225 controls, resulting in a sensitivity of 86% and a specificity of 90%, the test was validated in a prospective cohort of 150 healthy women who participated in a routine cervical screening program, 54 of whom developed EC within 3 years after sample collection.The sensitivity was substantially lower (52%), most likely because of the longterm storage of the specimen negatively affecting the signal.
Wever et al. showed an optimal panel of three DNA methylation markers with excellent diagnostic performance for detecting EC in a case-control study of 320 women with an AUC of 0.94 and 0.97 for cervicovaginal self-samples and cervical swabs, respectively. 31erzog et al. used cervical smears and vaginal swabs to evaluate DNA methylation in the genes GYPC and ZSCAN12 as predictors of EC. 34 The sensitivity and specificity in a case-control cohort of 137 women with cervical smears (71 EC patients) were 97.2% and 75.8%, respectively, and in another case-control cohort with 251 self-collected vaginal swabs (131 EC patients), they were 90.1% and 86.7%, respectively.In a cohort of 63 women with postmenopausal blood loss (eight EC patients), the vaginal swab test resulted in a sensitivity and specificity of 100% and 89.1%, respectively.They also performed a predictive assessment in 25 Lynch carriers to assess whether the test identifies not only women with cancer, but also women at risk of developing cancer because of genetic predisposition.Cervical smears performed during regular surveillance visits had a sensitivity of 33.3% and a specificity of 100% for detecting EC, with positive and negative predictive values of 50.7% and 92.2%, respectively.Another predictive assessment in a general population of 86 women of whom 32 developed EC, retrospective methylation analysis of cervical smear DNA collected 3 years prior to first diagnosis, revealed a sensitivity and specificity for diagnosing EC of 68.8% and 98.1%, respectively.The sensitivity was 90.8% when samples were analyzed up to 1 year prior to the EC diagnosis.
Wang et al. reported on cancer detection in a large cohort of controls (n = 712), OC patients (n = 245), EC patients (n = 382) and by mutation analysis of 18 genes using cervical smear DNA. 44They reported a sensitivity of 33% and 81% in OC and EC patients, respectively, with a specificity of 98.6%.

| Microbiome
Seven studies evaluated the microbiome in cervical smears of women with OC and EC (Table 2).Morikawa et al. and Nené et al. demonstrated that the composition of the microbiome differed between patients with OC and healthy controls. 50,52Morikawa et al. observed in a relatively small study cohort (n = 62) that a diversified, Lactobacilli-poor microbiome was associated with the presence of OC in premenopausal women. 50Nene et al. showed that the prevalence of women with non-Lactobacilli-dominated cervicovaginal microbiomes was higher both in patients with OC (n = 176) and in women with BRCA1 pathogenic variants (n = 109), compared with age-matched healthy women and women without BRCA1 pathogenic variants. 52 correlation between the occurrence of EC and a high vaginal pH in combination with the presence of some types of bacteria was observed in two other studies. 53,54

| Proteomics
A total of six studies evaluating proteomics in cervicovaginal specimen were included, from which no study included patients with a high-risk profile (Table 3).Rocconi et al. obtained cervicovaginal fluid samples for liquid chromatography-mass spectrometry (LC-MS) evaluation.Thirty peptides were detected with a statistical significance for detecting OC.They determined a combination of five proteins that were the best predictors for detecting OC with an AUC of 0.86. 55alis et al. and He et al. reported that the detection of CA-125 in cervicovaginal secretion could be used as a potential EC biomarker. 59,60In the study of Calis et al., the mean cervicovaginal secretion CA-125 levels in women with endometrial (pre) malignant pathology (n = 37) were significantly higher compared to those wo had other benign gynecological pathologies (n = 106). 59

| DISCUSSION
In this review, an overview of DNA methylation and mutation biomarkers, microbiome, and proteomic biomarkers that are detectable in cervicovaginal smears for the early detection of OC and EC is given.Most studies have studied the field of DNA analysis including both methylation and mutation.Studies generally used a case-control design and reported on OC and EC in the general population.Only a limited number of studies included women with a hereditary high-risk profile (i.e., BRCA1/2 pathogenic variant carriers, Lynch syndrome patients and/or PHTS patients).Determining DNA methylation biomarkers in cervicovaginal specimen seems to be the most promising technique for detecting OC and EC early in the general population, and this technique needs to be explored in women with a hereditary elevated risk of developing OC and EC.DNA methylation techniques have shown to be the most promising for detecting OC and EC, considering the large number of participants included and the relatively high accuracy of the tests.The large heterogeneity of the methylated genes used hampers a clear recommendation of the most relevant genes to detect OC and EC.In a cohort of 780 women, Barrett et al. analyzed CpGs methylation and found AUCs of 0.76 and 0.81 for OC and EC, respectively, based on a set of differentially methylated CpG sites. 33Herzog et al. detected EC with a panel of only two methylated genes (GYPC and ZSCAN12) with a sensitivity and specificity of >90% and 75%, respectively. 34This was the only study including Lynch patients resulting in a sensitivity and specificity for EC of 33.3% and 100%, respectively.This remarkable difference in diagnostic accuracy when compared to the general population could be explained by the relatively small number of included Lynch patients (n = 25).Wang et al. also showed promising results based on DNA mutation analysis; however, there was a large difference in the ages of EC patients and controls (62 and 34 years, respectively).Given that DNA mutations increase with age, it is difficult to judge whether the outcomes of the test are true discriminators. 61ith respect to the mutated genes in OC, the most found mutation is TP53.In this review, five studies reported on sensitivity of the presence of TP53 in OC.Sensitivities range.The sensitivities ranged between 33% and 64%. 27,38,39,44,62The mutated genes associated with EC are less consistent with multiple genes involved.Reijnen et al. reported on the most common mutations (n = 8) with different diagnostic values for detecting EC. 36 The studies in this review revealed a similar wide range of diagnostic accuracy of the mutated genes in women with EC, illustrating the challenges of using DNA mutations as a screening tool in both OC and EC. 27,36,37,41,44icrobiome studies that were performed in this review were generally explorative.3][54] However, these findings need to be interpreted with caution, as the mean age of the healthy control group in these studies was significantly lower than that of the cancer group, so the results may have been biased due to age-specific microbiome differences and menopausal status. 63However, in research on cervical precancerous lesions, differences in the cervical microbiome have also been associated with a higher incidence of cervical precancerous lesions. 64,65Further work is needed to investigate whether OC-specific and ECspecific microbiomes can be identified.
The field of cancer studies has seen a rise in the adoption of proteomics approaches.The use of proteomicsbased technologies such as LC-MS has allowed the discovery of potential biomarkers and patterns of protein expression.Indeed, the genetic and epigenetic changes occurring at the onset of cancer also logically manifest at the protein level; therefore, proteomics is a potentially effective technique.Based on the small sample size and lack of matched controls in published studies, it remains unclear whether proteomics might contribute to the early detection of OC and EC.
Despite including RNA techniques in our search strategy, no articles that used this technique as a diagnostic or screening tool could be identified.Transcriptomics seems to be a yet unexplored field in the detection of OC and EC, although it has been used in the detection of cervical neoplasia.Cervical scrapes can be used for high-throughput targeted next-generation RNA sequencing in which human gene expression profiles (with simultaneous detection of genetic mutations in nonsilenced genes) and microbial gene expression profiles can be simultaneously analyzed via high-throughput methods. 64,66,67Further combining these tests with methylation analysis may be worthwhile for women predisposed to OC or EC because of hereditary cancer syndromes.
Overall, the detection rates of EC are greater than the detection rates of OC.This finding can be explained by the diagnostic target organ, the cervix, being in closer proximity to the uterine cavity than to the ovaries and fallopian tubes.This hypothesis is strengthened by the study of Wang et al. compared the detection rate of OC and EC using a cervical smear and a brush inserted into the uterine cavity (Tao brush) in favor of the Tao brush. 44aritschnegg et al. investigated an approach for lavage of the uterine cavity for the early detection of OC. 68 They demonstrated that cells originating from ovarian tumors are shed and can be retrieved by washing out the uterine cavity.This study successfully detected OC and EC, as well as asymptomatic and clinically undetectable (low CA-125 and normal ultrasound) OC in a BRCA1 pathogenic variant carrier, suggesting that this method is a promising tool for the early detection of OC.Since this intra-uterine lavage technique is more invasive and less patient friendly than a cervicovaginal smear, this was beyond the scope of the current review.However, these findings support the hypothesis that tumor cells shed from the fallopian tube might enter the uterine cavity and may also be detected in cervicovaginal smears.This systematic review is the first to provide a clear overview of the various methods and techniques used for the early detection of OC and EC using cervicovaginal smear biomarkers.These data are highly valuable for guiding future studies to further explore techniques and improve the diagnostic accuracy of early diagnosis of OC and EC.Whether these techniques are suitable for highrisk populations is still unclear due to the low number of studies including high-risk subgroups.Only five studies reported on the detection of OC and EC in high-risk profile groups, of which only one reported on Lynch syndrome (n = 25). 27,29,34,44,45ome further limitations need to be addressed.High heterogeneity was found between studies with a wide range of analyzed biomarkers.This makes it difficult to compare the detection rates of the analyzed genes.Due to this heterogeneity, it was unfortunately not possible to perform a metaanalysis including all studies separately for OC and EC.The study populations were relatively small, with only eight studies reporting on more than 150 patients.Studies differ in including asymptomatic and symptomatic patients.Moreover, for future clinical implementation, the distinction between screening or early detection is relevant and requires the alignment of inclusion criteria in future studies that might be different for EC (postmenopausal bleeding/ germline mutations) and OC (germline mutations).
In summary, there is a need for a more patient-friendly and easily accessible technique to detect OC and EC.This review shows that DNA methylation panel analyses of cervicovaginal smears are feasible techniques as a minimally invasive tool for the early detection of OC and EC in the general population.The future perspectives regarding detection through microbiome and proteomics approaches are currently unclear because of limited data.Implementation in the daily gynecological clinic is hampered by the lack of large prospective validation studies, which are needed due to the large heterogeneity in published studies.In addition, evaluation of cervicovaginal DNA methylation techniques in a hereditary high-risk population is required to determine the benefit in this specific group of women with increased risk of developing OC and EC, who might benefit most from these innovative approaches.

E 1
Preferred Reporting Items for Systematic Reviews and Meta-Analyses flow diagram of the selection of studies to be included in this review.

Identification of studies via databases Identification Screening Included T A B L E 1
Overview of studies using DNA methylation and/or mutation analysis techniques on cervicovaginal specimens to detect OC and EC.

Author, year Study design Biospecimen Tumor type N total N OC N EC N control High-risk profile group Methodology of testing Methylated or mutated gene Findings
Abbreviations: AUC, area under the curve; ddPCR, droplet digital polymerase chain reaction; EC, endometrial cancer; NGS, next-generation sequencing; OC, ovarian cancer; qMSP, quantitative methylation-specific PCR; smMIP, single-molecule molecular inversion probes; WGS, whole-genome sequencing.
Overview of studies using the cervicovaginal microbiome to detect OC and EC.Overview of studies using cervicovaginal proteomics to detect OC and EC.
T A B L E 2 T A B L E 3Abbreviations: AUC, area under the curve; EC, endometrial cancer; EIN, endometrial intraepithelial neoplasia; OC, ovarian cancer.